Project Summary: The long-term objective of this proposal is to understand the molecular events that lead to the determination and differentiation of different retinal cell types. During mammalian retinogenesis, seven classes of cells are specified from multipotent progenitors by the action of various intrinsic and extrinsic factors. Recent molecular genetic studies involving loss-of-function and gain-of-function approaches have uncovered a number of transcription factors as pivotal intrinsic regulators of retinogenesis. These factors are found to act at different developmental processes to establish progenitor multipotency, define progenitor competence, determine cell fates, and/or specify cell types and subtypes. Therefore, transcription factors play key roles in controlling cell specification and differentiation during retinogenesis. Despite these important advances, however, the molecular targets and signaling events downstream from many transcription factors involved in retinal development still remain poorly understood. In this application, experiments are proposed that will focus on the molecular and developmental events regulated by the Foxn4 winged helix/forkhead transcription factor and its downstream molecules. Our previous studies have demonstrated a crucial role for Foxn4 in competence acquisition and/or fate commitment to amacrine and horizontal cells. However, it is unclear at present what are the genes and signaling events that act genetically downstream of Foxn4 to mediate its critical functions. To address these issues, four specific aims will be pursued: i) to analyze the role of Foxn4 in the specification of photoreceptor cells. Utilizing molecular, cellular and overexpression approaches, we aim to investigate in Foxn4 null retinas the expression profiles of photoreceptor and ganglion regulator genes, and the generation and distribution patterns of photoreceptor cells, as well as to examine the effect of misexpressed Foxn4 on the differentiation of photoreceptor cells;ii) to investigate Dll4-Notch signaling events during retinogenesis by overexpression analysis. We aim to employ an overexpression approach to determine the effect of Dll4 and its dominant-negative form on retinal cell specification and differentiation, and to investigate by chromatin immunoprecipitation and electrophoretic mobility shift assays whether Foxn4 has the ability to directly bind and activate the Dll4 promoter;iii) to study the biological function of Dll4 during retinogenesis by conditional gene targeting. We aim to generate and characterize retina-specific Dll4 knockout mice to identify the developmental and cellular processes that Dll4-Notch signaling regulates and establish a functional relationship between Dll4 and Foxn4 during mammalian retinogenesis;and iv) to map the fate of Foxn4- expressing retinal progenitors. Our goal is to determine the lineages of Foxn4-expressing retinal progenitors using the Cre-loxP fate-mapping strategy. The proposed studies together are expected to provide important insights into the genetic regulatory networks involved in retinal cell specification and differentiation.